Cell surface serine proteases as regulators of epithelial development, homeostasis regeneration, and malignancy (33% effort) Background: Cell behavior in higher eukaryotes is regulated by a large number of proteases and protease inhibitors that operate in the pericellular environment to provide focal proteolysis that is essential for cytokine/growth factor maturation, matrix remodeling, signaling receptor activation and shedding, ion channel activity, and more. We are continuing our efforts to understand the molecular functions of membrane-anchored serine proteases and their inhibitors in vertebrate development, epithelial homeostasis, and epithelial carcinogenesis by using a combined biochemical, cell biological, and genetic approach. Research accomplished: Matriptase zymogen supports epithelial development, homeostasis and regeneration Trypsin-like serine proteases generally are synthesized as inactive pro-enzymes (zymogens) with an N-terminal extension that spatially distorts the active site to render the protease catalytically inactive. Conversion to the active protease (zymogen conversion) occurs by removal of the N-terminal extension by a single proteolytic cleavage after an Arg or Lys residue, which is located within a highly conserved activation cleavage site. Removal of this N-terminal extension spatially reorients the catalytic domain to its active conformation. We and other have previously shown that matriptase is unusual among trypsin-like serine proteases in that the zymogen possesses appreciable enzymatic activity. Two hypotheses have been advanced regarding this observation: a) The intrinsic activity of the matriptase zymogen serves exclusively to mediate matriptase zymogen conversion through auto-activation. b) The matriptase zymogen is a biologically-active molecule that is capable of cleaving heterologous proteins to execute essential biological functions of the protein. To test these hypotheses, we used gain and loss of function genetics in mice. Unexpectedly, transgenic mice mis-expressing a zymogen-locked version of matriptase in the epidermis displayed pathologies previously reported for transgenic mice mis-expressing wildtype epidermal matriptase. Equally surprising, mice engineered to express only zymogen-locked endogenous matriptase, unlike matriptase null mice, were viable, developed epithelial barrier function, and regenerated injured epithelium. Taken together, these findings show that the matriptase zymogen is biologically active and is capable of executing developmental and homeostatic functions of the protease. Endocytic extracellular matrix degradation in physiological and pathophysiological processes Background: Research performed within the last five decades led to the identification and extensive characterization of extracellular matrix (ECM)-degrading enzymes. However, the cellular orchestration of ECM degradation and the contribution of endocytic pathways and endocytic receptors to ECM turnover is less studied and understood. Research accomplished: Identification, isolation, and characterization of collagen-degrading macrophages in tumors We recently discovered a key function of mannose receptor (MR)-dependent endocytic collagen degradation by M2-polarized macrophages in collagen clearance from non-neoplastic tissues. Although collagen degradation in the context of malignant tumor progression has been hypothesized to be executed directly by the malignant cells or by tumor-associated fibroblasts, this raised the possibility that macrophages could be involved in tumor-mediated extracellular matrix turnover. In collaboration with Roberto Weigert, NCI, by using mice with genetically-encoded fluorescently-labeled fibroblasts, tumor cell lines, and macrophages, in combination with whole mount immunostaining, we have been able to visualize and quantify endocytic collagen degradation by these three principal cellular constituents of tumors in vivo. Unexpectedly, all detectable collagen uptake by tumors was observed in a subpopulation of cells identified as macrophages by their expression of CD11b, CD45, and F4/80, irrespectively of whether the tumor cells were of epithelial, mesenchymal or neural crest origin. Furthermore, these cells expressed MR, and internalized collagen in a MR-dependent manner. In support of this pivotal role of macrophages in tumor-associated collagen turnover, comparative whole-genome transcriptomic profiling of FACS-isolated tumor cells, tumor-associated fibroblasts, and tumor-associated macrophages showed high collagenase expression by macrophages and fibroblasts, and a low collagenase expression by tumor cells. Furthermore, lineage-ablation studies revealed that collagen-degrading, tumor-associated macrophages predominantly originated from circulating CCR2+ inflammatory monocytes. The study identifies a novel function of tumor-associated macrophages in facilitating tumor progression through endocytic collagen turnover and, furthermore, establishes blood-derived macrophages as a central cell type engaged in tumor-associated collagen degradation. Reengineered bacterial cytotoxins as anti-tumor and protease imaging agents (33% effort) Background: Elevated expression of matrix-degrading proteases is a hallmark of human cancer. We are engaged in a long-standing collaboration with Steve Leppla, NIAID, on the development of reengineered bacterial cytotoxins, activated by proteases expressed in the tumor microenvironment, as novel therapeutic agents for cancer and as tools for the imaging of specific cell surface proteolytic activity. Research accomplished: Development of an anthrax toxin protective antigen variant that selectively utilizes the CMG2 receptor for cellular uptake and tumor targeting Our research described above identified CMG2 as the principal receptor mediating the anti-tumor activity of our modified anthrax toxins. This raised the possibility that anthrax toxin protective antigen (PrAg) variants that selectively bound CMG2 could have an improved therapeutic index by eliminating pathology associated with cellular intoxication via TEM-8. Affinity selection of PrAg mutants with random mutations in the receptor binding domain revealed that residue I656 played a critical role in PrAg binding to TEM8, but to a much lesser degree binding to CMG2. Comparative analysis of PrAg variants with substitution of this residue with the 19other available amino acids identified PrAg I656Q as a highly CMG2 receptor-selective PrAg variant. When furnished with a matrix metalloproteinase (MMP)-selective activation cleavage site, the PrAg I656Q variant displayed potent anti-tumor activity towards xenografted tumors in mice, while demonstrating low systemic toxicity. The study identifies additional avenues for improvement of the toxicity profile of protease-activated anthrax toxins.